Conjugates of anticoagulant and protein

ABSTRACT

Conjugates are provided which are covalently bonded conjugates of an anticoagulant and protein that are prepared in the presence of a coupling agent that forms amide linkages between the anticoagulant and the protein. Such amide linking coupling agents exclude highly toxic coupling agents such as CNBr. These conjugates are useful for enhancing the blood compatibility of certain surfaces of a prosthetic device, a surgical apparatus, or an extra-corporeal medical device.

This is a division of application Ser. No. 716,197, filed Mar. 26, 1985,now U.S. Pat. No. 4,634,762, which is a division of application Ser. No.449,248, filed Dec. 13, 1982, now U.S. Pat. No. 4,526,714.

BACKGROUND AND DESCRIPTION OF THE INVENTION

The invention relates to a process for the preparation of a conjugate ofan anticoagulant such as heparin to a water soluble protein, typicallyhuman protein, by coupling the heparin to the protein in an aqueousmedium and in the presence of a coupling agent.

In the article "Heparin Coupled to Albumin, Dextran and Ficoll;Influence on Blood Coagulation and Platelets and in vivo Duration",Thrombosis Research 7 (1975) 273-284, A. N. Teien et al, the authorsreport upon an investigation carried ut by them which indicates that aheparin-albumin complex possesses anti-coagulation and aggregationeffects equal to those of free heparin. The heparain-albumin complexinvolved in their investigation was prepared by adding CNBr to asolution containing dextran, effecting an activation of the dextran inorder to bridge heparin to the albumin, after which the whole is mixedwith an aqueous solution of heparin and albumin. The heparin-albumincomplexes so obtained are then lyophilized and stored in Veronal bufferand are incorporated in a stock solution containing 16 mg heparin percm³, which assumes complete recovery of heparin. The complexes thusformed possess a molecular weight distribution due to, inter alia, theheterogenity of the commercial heparin preparations, which also exhibita distribution of molecular weights. Using CNBr in such a process isundesirable because of the very high toxicity of CNBr. Also, using CNBrin this manner does not form covalent amide linkages.

An object of the invention is an improved process for the preparation ofconjugates of heparin to a human protein, such conjugates being suitableto treat a material surface or substrate for the improvement of itsblood compatibility, particularly regarding the prevention of bloodcoagulation and damage of blood components. When a substrate is coatedwith such a conjugate, its blood compatibility is improved, and theconjugates of this invention are advantageously applied to artificialorgans, implants, catheters, and other medical prostheses which arecontacted with blood.

According to this invention, the coupling agent for forming theconjugates should form amide linkages between an anticoagulant such asheparin and protein. The preferred coupling agent in this regard is1-ethyl-3-dimethylaminopropyl carbodiimide (or EDC).

EDC has been found to provide the best and most reproducible results,particularly when EDC is applied as the coupling agent and when thecoupling is carried out in an aqueous solution of heparin and theprotein. Within the process according to the invention, the couplingreaction between the carboxyl groups present in heparin and --NH₂-groups of lysine residues present in the protein, is a direct couplingbetween these groups, a heparin intermediate product being formedwherein the carboxyl groups are activated by EDC to active ester groups,which activated carboxyl groups are in turn reactive towards the aminogroups of the protein.

In a further elaboration of the process according to the invention thatutilizes EDC, the heparin and the protein are each provided dissolved inwater; the pH of the solution is adjusted to a value of not higher than5.5, preferably between about 5.0 and 5.5; an aqueous solution of EDC isadded batchwise while maintaining the pH on a level of not higher than5.5; the coupling reaction is carried out in the reaction mixture whileagitating it; and the heparin-protein conjugate is recovered from thereaction mixture.

Normally, the reaction is carried out at room temperature. However,higher temperatures may be applied, for example body temperature, and upto the denaturation temperature of the applied protein, typically up toabout 60 degrees centigrade. Generally, the concentration of thecomponents heparin and protein to be coupled can be varied greatly inthe aqueous solutions, and the ratio of these components to each otherin the conjugate may be varied up to a certain level by varying theweight ratio of heparin to protein.

With further reference to the pH values of this process, when thecoupling reaction according to this invention uses EDC, if there is afailure to adjust and maintain the pH-value at or below a pH value ofabout 5.5, the rate of the reaction of heparin with EDC is very lowbecause the concentration of the protonated carbodiimide groups of EDCis too low. Coupling agents other than EDC may exhibit different upperpH limits.

If the pH is too low, there are two undesirable results; denaturation ofthe protein takes place, and the sulphamate groups in the heparin willbe transformed into amino groups. Therefore, it is preferred that the pHof the reaction mixture is adjusted to and maintained at a value that isequal to or greater than 5.0.

When heparin-protein conjugates are formed according to the invention,the composition of the components to be coupled may be at various weightratios during the coupling reaction. A typical preferred weight ratiofor the amounts of heparin and protein used in the proces is such thatthe mol ratio of heparin to protein in the conjugate is about 1 to 1,although other weight ratios may be utilized so that heparin-proteinconjugates are obtained at a different mol ratio. One characteristic tobe avoided in this regard is to attempt to couple excessive heparinmolecules onto the protein to the extent that the conjugate is overlyhydrophilic and will not adsorb onto hydrophobic substrates. Heparindoes not readily adsorb onto hydrophobic substrates, while a proteinsuch as albumin does so readily adsorb, and typically a mol ratio ofheparin to albumin above 3 to 1 should be avoided.

Regarding the protein which is to be used as the reaction component, andwhich is a generally water soluble human protein, such may be a watersoluble protein, suitable for coupling and appearing in blood, forexample albumin, fibrinogen, γ-globulin, and the like. Albumin ispreferred because, of the human proteins, albumin has the highestconcentrations in human blood. Moreover, albumin exerts a strengthenedinhibiting effect relative to the adhesion of blood platelets. Theseproteins may be used as such or in crosslinked form, for example bycrosslinking with an aldehyde, for example glutaraldehyde. Dependent onthe protein component used, one should always experimentally determinethe desired pH of the reaction mixture, particularly from the point ofview of avoiding undesirable denaturation of the particular proteinused.

With regard to the anticoagulant, for example, heparin fractions may beused which are separated from heparin by fractionating it on the basisof difference in molecular weight and/or affinity relative toanti-thrombin III. Because it is generally known that, by fractionatingheparin with immobilized anti-thrombin III, heparin fractions with highand low affinity may be obtained, the use of fractionated heparin offersthe possibility to control the anti-thrombogenic activity of theheparin-protein conjugate.

After the coupling reaction has taken place, the reaction mixturecontains not only the desired covalently bonded heparin-proteinconjugate, but also free, that is to say non-reacted, protein andheparin. To isolate the conjugate from the reaction mixture, afteroptionally having subjected the mixture to a prepurification, for examleby dialysing it to remove superfluous EDC, the mixture preferably ispassed over a first sorption agent, which sorbs the heparin-proteinconjugate, free protein and free heparin. Whereupon, by elution witheluent of increasing ionic strength, it is possible to first desorb thefree protein and subsequently the heparinous compounds, and the eluatewhich contain the heparinous compounds are preferably then passed over asecond sorption agent, by elution of which one separately desorbs thefree heparin and the heparin-protein conjugate, respectively, andisolates the heparin-protein conjugate. Preferably such a first sorptionagent would be an anion exchange ssynthetic resin material, and thesecond sorption agent would be a material with different affinityrelative to the heparinous compounds.

The invention also relates to a process for the improvement of the bloodcompatibility of a material surface or substrate by coating it withheparin or with heparin analogues as is generally discussed in"Artificial Organs, Proceedings of a Seminar on the ClinicalApplications of Membrane Oxygenators and Sorbent Based Systems",MacMillan Press Ltd., 1977. In chapter 26 thereof, at pages 235-247, J.Feijen under the title "Thrombogenisis Caused by Blood-Foreign SurfaceInteraction" discusses the developments in the field of the bloodcompatibility of material surfaces, particularly regarding the occurringprotein adsorption, adhesion of blood platelets and activation ofintrinsic coagulation.

Within the framework of improving the blood compatibility of a materialsurface by coating it with an anticoagulant such as heparin or heparinanalogues, the invention is characterized in that aheparin/non-crosslinked conjugate, prepared by the process according tothe invention described hereinabove, is adsorbed to a hydrophobic orhydrophilic material surface, particularly to a hydrophobic materialsurface, in which case the protein moiety will be directed to thematerial surface and the heparin moiety in the blood.

In some applications and aspects of this invention, it is desired tominimize the desorption of the coated anticoagulant-protein conjugate byexchange with other plasma proteins. Such can be accomplished bycross-linking the protein moiety of the conjugate, typically after theconjugate has been formed and coated onto the material surface orsubstrate.

Apart from adsorption for the improvement of the blood compatability ofa material surface by coating it with heparin or heparin analogues, itis also possible to prepare and provide anticoagulant protein conjugatesaccording to the present invention which are chemically attached to thematerial surface or substrate. In this instance, the protein isconsidered as a so-called "spacer" group, while it is also possible toeffect an eventual crosslink-reaction of the protein moiety after thematerial surface is coated with the heparin-protein conjugate having anon-crosslinked protein moiety.

The improvement of the blood compatability of a material surface bycoating it with an anticoagulant such as heparin or heparin analoguesmay also be effected by providing the material surface with acrosslinked protein layer, whereupon heparin is coupled to the proteinby the application of EDC as the coupling agent. In the coating obtainedwith this embodiment of the invention, the coupling takes place via theamino groups of the protein, and the protein may also be considered as a"spacer" group between the material surface and the heparin.

Depending upon the particular aspect of this invention needed to providea desired result, the conjugates may be generally firmly attached to thesubstrate surface, usually in association with a cross-linkingoperation, or they may be only adsorbed thereto. Adsorbed conjugateswill exhibit desorption to the extent that the conjugates will becomegenerally released such that the conjugates will provide anticoagulationproperties to the blood flowing past the substrate. In effect, thisaspect of the invention provides controlled release of anticoagulantproperties, which can be valuable in connection with treatments usingextra-corporeal devices such as catheters, in connection withpretreatment of prosthetic devices such as artificial blood vesselswhere enhanced tissue ingrowth is very advantageous, or in connectionwith surgical apparatus such as oxygenators where only temporaryanticoagulation is needed. The rate and extent of such desorption orcontrolled release of conjugates will depend upon the particularconjugate, upon the hydrophobic or hydrophilic nature of the substratebeing coated, and upon the rate of blood flow that contacts thesubstrate.

The invention will be further explained by the following examples, inwhich albumin is applied as the protein component.

EXAMPLE I

(a) Preparation

In this example, the process is carried out at room temperature. 770 mgof heparin and 2590 mg of human serumalbumin were dissolved in 39 cm³ ofwater. By means of 1.0N HCl, the pH of this solution was adjusted tobetween 5.0 and 5.5. During the addition, a white-flaky precipitate isgenerated, which is dissolved after about 30 minutes. Thereupon,incremental batches of 1 cm³ EDC-solution (concentration 32.5 mg/cm³)were added. Eight incremental batches were added at 30-minute intervals.As necessary, the pH was maintained at between 5.0 and 5.5 by theaddition of 1.0N HCl or 1.0N NaOH. After all of the EDC was added, thepH of the solution was adjusted to 7.5 by means of 1.0N NaOH, whereuponthe solution was agitated during 20 hours at room temperature.Thereafter, the solution was dialyzed for 2 hours against a 25 mmoltris/HCl solution (pH 7.5) to remove superfluous EDC.

(b) Isolation of the conjugate

In addition to the desired albumin-heparin conjugate, the reactionmixture also contained albumin and heparin which had not yet reacted.For the separation of these free components, use was made of an anionexchange column and an affinity column, i.e. a column provided withdiethylaminoethyl-cellulose (DEAE-cellulose), followed by a columnprovided with Cibracron Blue Sepharose (CB-Sepharose). The dialyzedreaction mixture obtained in this manner was passed over aDEAE-cellulose column (5 mg material/cm³ gel) which was in equilibriumwith 25 mmol tris/HCl at pH=7.5. Then, the column was eluted with onecolumn volume of 25 mmol tris/HCl (pH 7.5). Thereupon the column waseluted with 1.5-2 times the column volume 150 mmol NaCl+25 mmol tris/HCl(pH 7.5). At this step the ionic strength of the elution liquid was suchthat the "free" albumin which had not reacted was being separated fromthe column and eluted. Next, the DEAE-column was eluted with 1.5-2 timesthe column volume 500 mmol NaCl+25 mmol tris/HCl (pH 7.5). At this ionicstrength the heparin-albumin conjugate as well as the non-reacted "free"heparin was separated from the column. The fractions which were receivedthrough elution of the column with the solution containing 500 mmolNaCl, and which had an optical density at 280 nm (λ_(max) albumin)>0.2,were combined and used for further purification with CB-Sepharose. Theelution pattern of the DEAE-separation, as described above, is shown inFIG. 1. The CB-Sepharose column was equilibrated with a 1% NaCl and 25mmol tris/HCl (pH 7.5) solution, whereupon the combined fractions of theDEAE-column were passed over the CB-Sepharose column. To remove the"free" heparin present in the solution, the column was eluted with 1times the column volume 1% NaCl and 25 mmol tris/HCl (pH 7.5) solution.Thereupon, the albumin-heparin conjugate was eluted from the CB-columnby means of a 1% NaCl+25 mmol tris/HCl (pH 7.5)+250 mmol KSCN solution.The fractions having an optical density at 280 nm>0.2 were combined,exhaustively dialyzed against water and finally freeze dried. Theelution pattern of a CB-Sepharose separation as described above is shownin FIG. 2. All chromatographic experiments were carried out at atemperature of 4° C.

EXAMPLE II Anti-thrombogenic activity of heparin-albumin conjugates

A heparin-albumin conjugate was prepared using the process described inExample I, starting with nonfractionated heparin.

The conjugate prepared according to Example I was passed over ananti-thrombin III Sepharose-column. The elution pattern of thisconjugate showed two fractions (FIG. 3); both fractions were measured onthrombin-inactivation (FIG. 4) and on factor Xa inactivation (FIG. 5).The results show that the several fractions differ in anti-coagulantactivity.

Moreover, the conjugate prepared according to Example I, on the basis ofboth inactivation experiments, was measured and compared with heparin,which was used for the synthesis of the heparin-albumin conjugate. Theresults thereof in FIG. 6 and in FIG. 7 show that the conjugate hasworking properties comparable to those of the heparin that was used forthe synthesis of the conjugate.

We claim:
 1. An article including a surface having an enhanced bloodcompatibility coating, wherein the coating is effected from a conjugateof anticoagulant and a water soluble protein, said protein being anon-crosslinked protein or a crosslinked protein, said conjugate havingbeen formed by coupling a protein component and an anticoagulant with anamide bond forming agent.
 2. The article according to claim 1, whereinthe coating is effected from a conjugate of heparinous material and awater soluble human protein, and wherein said amide bond forming agentis 1-ethyl-3-dimethylaminopropyl carbodiimide.